Lungs Of Transgenic Mice Research Articles

Porcine \u03b2-defensin 2 inhibits proliferation of pseudorabies virus in vitro and in transgenic mice

BackgroundPorcine β-defensin 2 (PBD-2), produced by host cells, is an antimicrobial cysteine-rich cationic peptide with multi-functions. Previous studies have demonstrated that PBD-2 can kill various bacteria, regulate host immune responses and promote growth of piglets. However, the antiviral role of PBD-2 is rarely investigated. This study aimed to reveal the antiviral ability of PBD-2 against pseudorabies virus (PRV), the causative pathogen of Aujeszky’s disease, in PK-15 cells and in a PBD-2 expressing transgenic (TG) mouse model.MethodsIn this study, the cytotoxicity of PBD-2 on PK-15 cells was measured by CCK-8 assay. PK-15 cells were incubated with PRV pre-treated with different concentrations of PBD-2 and PRV titers in cell culture supernatants were determined by real-time quantitative PCR (RT-qPCR). TG mice and wild-type (WT) mice were intraperitoneally injected with PRV and the survival rate was recorded for 10 days. Meanwhile, tissue lesions in brain, spleen and liver of infected mice were observed and the viral loads of PRV in brain, liver and lung were analyzed by RT-qPCR.ResultsPBD-2 at a maximum concentration of 80 μg/mL displayed no significant cytotoxicity on PK-15 cells. A threshold concentration of PBD-2 at 40 μg/mL was required to inhibit PRV proliferation in PK-15 cells. The survival rate in PBD-2 TG mice was 50% higher than that of WT mice. In addition, TG mice showed alleviated tissue lesions in brain, spleen and liver compared with their WT littermates after PRV challenge, while viral loads of PRV in brain, liver and lung of TG mice were significantly lower than that of WT mice.ConclusionsPBD-2 could inhibit PRV proliferation in PK-15 cells and protect mice from PRV infection, which confirmed the antiviral ability of PBD-2 both in vitro and in vivo. The application of PBD-2 in developing anti-viral drugs or disease-resistant animals can be further investigated.

Platelet Extracellular Vesicles Drive Inflammasome-IL-1β-Dependent Lung Injury in Sickle Cell Disease.

Rationale: Intraerythrocytic polymerization of Hb S promotes hemolysis and vasoocclusive events in the microvasculature of patients with sickle cell disease (SCD). Although platelet-neutrophil aggregate-dependent vasoocclusion is known to occur in the lung and contribute to acute chest syndrome, the etiological mechanisms that trigger acute chest syndrome are largely unknown.Objectives: To identify the innate immune mechanism that promotes platelet-neutrophil aggregate-dependent lung vasoocclusion and injury in SCD.Methods:In vivo imaging of the lung in transgenic humanized SCD mice and in vitro imaging of SCD patient blood flowing through a microfluidic system was performed. SCD mice were systemically challenged with nanogram quantities of LPS to trigger lung vasoocclusion.Measurements and Main Results: Platelet-inflammasome activation led to generation of IL-1β and caspase-1-carrying platelet extracellular vesicles (EVs) that bind to neutrophils and promote platelet-neutrophil aggregation in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. The inflammasome activation, platelet EV generation, and platelet-neutrophil aggregation were enhanced by the presence of LPS at a nanogram dose in SCD but not control human blood. Inhibition of the inflammasome effector caspase-1 or IL-1β pathway attenuated platelet EV generation, prevented platelet-neutrophil aggregation, and restored microvascular blood flow in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro.Conclusions: These results are the first to identify that platelet-inflammasome-dependent shedding of IL-1β and caspase-1-carrying platelet EVs promote lung vasoocclusion in SCD. The current findings also highlight the therapeutic potential of targeting the platelet-inflammasome-dependent innate immune pathway to prevent acute chest syndrome.

Loss of LRP1 promotes acquisition of contractile-myofibroblast phenotype and release of active TGF-β1 from ECM stores

In healing tissue, fibroblasts differentiate to α-smooth muscle actin (SMA)-expressing contractile-myofibroblasts, which pull the wound edges together ensuring proper tissue repair. Uncontrolled expansion of the myofibroblast population may, however, lead to excessive tissue scarring and finally to organ dysfunction. Here, we demonstrate that the loss of low-density lipoprotein receptor-related protein (LRP) 1 overactivates the JNK1/2-c-Jun-Fra-2 signaling pathway leading to the induction of α-SMA and periostin expression in human lung fibroblasts (hLF). These changes are accompanied by increased contractility of the cells and the integrin- and protease-dependent release of active transforming growth factor (TGF)-β1 from the extracellular matrix (ECM) stores. Liberation of active TGF-β1 from the ECM further enhances α-SMA and periostin expression thus accelerating the phenotypic switch of hLF. Global gene expression profiling of LRP1-depleted hLF revealed that the loss of LRP1 affects cytoskeleton reorganization, cell-ECM contacts, and ECM production. In line with these findings, fibrotic changes in the skin and lung of Fra-2 transgenic mice were associated with LRP1 depletion and c-Jun overexpression. Altogether, our results suggest that dysregulation of LRP1 expression in fibroblasts in healing tissue may lead to the unrestrained expansion of contractile myofibroblasts and thereby to fibrosis development. Further studies identifying molecules, which regulate LRP1 expression, may provide new therapeutic options for largely untreatable human fibrotic diseases.

Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality.

The circadian clock is important for cellular and organ function. However, its function in sickle cell disease (SCD), a life-threatening hemolytic disorder, remains unknown. Here, we performed an unbiased microarray screen, which revealed significantly altered expression of circadian rhythmic genes, inflammatory response genes, and iron metabolic genes in SCD Berkeley transgenic mouse lungs compared with controls. Given the vital role of period 2 (Per2) in the core clock and the unrecognized role of Per2 in SCD, we transplanted the bone marrow (BM) of SCD mice to Per2Luciferase mice, which revealed that Per2 expression was up-regulated in SCD mouse lung. Next, we transplanted the BM of SCD mice to period 1 (Per1)/Per2 double deficient [Per1/Per2 double knockout (dKO)] and wild-type mice, respectively. We discovered that Per1/Per2 dKO mice transplanted with SCD BM (SCD → Per1/Per2 dKO) displayed severe irradiation sensitivity and were more susceptible to an early death. Although we observed an increase of peripheral inflammatory cells, we did not detect differences in erythrocyte sickling. However, there was further lung damage due to elevated pulmonary congestion, inflammatory cell infiltration, iron overload, and secretion of IL-6 in lavage fluid. Overall, we demonstrate that Per1/Per2 is beneficial to counteract elevated systemic inflammation, lung tissue inflammation, and iron overload in SCD.-Adebiyi, M. G., Zhao, Z., Ye, Y., Manalo, J., Hong, Y., Lee, C. C., Xian, W., McKeon, F., Culp-Hill, R., D' Alessandro, A., Kellems, R. E., Yoo, S.-H., Han, L., Xia, Y. Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality.

Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization.

To investigate the systemic sclerosis-related phenotype in fos-related antigen-1 transgenic mice and its underlying mechanisms. Lung and skin sections of constitutive fos-related antigen-1 transgenic mice and wild-type mice were examined by tissue staining and immunohistochemistry. The tricuspid regurgitation pressure gradient was measured by transthoracic echocardiography with a Doppler technique. To assess the impact of fos-related antigen-1 expression on macrophage function, bone marrow-derived mononuclear cells were derived from mice that expressed fos-related antigen-1 under the control of doxycycline and wild-type littermates. These bone marrow-derived mononuclear cells were induced to differentiate into macrophages with or without doxycycline, and analyzed for gene and protein expression. Finally, lung explants obtained from systemic sclerosis patients and control donors were subjected to immunohistochemistry. The lungs of fos-related antigen-1 transgenic mice showed excessive fibrosis of the interstitium and thickening of vessel walls, with narrowing lumen, in an age-dependent manner. The tricuspid regurgitation pressure gradient was significantly elevated in fos-related antigen-1 transgenic versus control mice. Increased dermal thickness and the loss of subdermal adipose tissue were also observed in the fos-related antigen-1 transgenic mice. These changes were preceded by a perivascular infiltration of mononuclear cells, predominantly consisting of alternatively activated or M2 macrophages. Overexpressing fos-related antigen-1 in bone marrow-derived mononuclear cell cultures increased the expression of M2-related genes, such as Il10, Alox15, and Arg1. Finally, fos-related antigen-1-expressing M2 macrophages were increased in the lung tissues of systemic sclerosis patients. The fos-related antigen-1 transgenic mouse serves as a genetic model of systemic sclerosis that recapitulates the major vascular and fibrotic manifestations of the lungs and skin in systemic sclerosis patients. M2 polarization mediated by the up-regulation of fos-related antigen-1 may play a critical role in the development of systemic sclerosis.

HIV-1 Protein gp120 Induces Mouse Lung Fibroblast-to-Myofibroblast Transdifferentiation via CXCR4 Activation

BackgroundIndividuals with HIV have ∼2-fold increased risk of developing pulmonary fibrosis. The mechanism(s) by which this occurs has yet to be determined. HIV-1 protein gp120 activates CXCR4 in the lymphocyte, promoting a variety of intracellular signaling pathways including those common to TGFβ1 associated with lung fibroblast-to-myofibroblast transdifferentiation. We hypothesized that gp120 promotes pulmonary fibrotic changes via activation of CXCR4 in the lung fibroblast. MethodsMouse primary lung fibroblasts (PLFs) were cultured ± gp120, then analyzed for α-SMA expression and stress fiber formation. In parallel, PLFs were cultured ± gp120 ± AMD3100 (a CXCR4 antagonist), and α-SMA, pan and phospho-Akt, and total and phospho-MAPK (or ERK1/2) protein expression was quantified. Finally, lungs and PLFs from wild-type and HIV-1 transgenic mice were analyzed for hydroxyproline and α-SMA content. Resultsgp120 treatment increased α-SMA expression and myofibroblast differentiation in PLFs. gp120 treatment activated phosphorylation of ERK1/2, but not PI3K-Akt. Pretreatment with AMD3100 inhibited gp120-induced ERK1/2 phosphorylation and gp120-induced α-SMA expression. In parallel, there was a significant increase in hydroxyproline content in lungs from older HIV-1 transgenic mice and a >3-fold increase in α-SMA expression in PLFs isolated from HIV-1 transgenic mice. Conclusionsgp120 induces α-SMA expression and fibroblast-to-myofibroblast transdifferentiation by activating the CXCR4-ERK1/2 signaling pathway in mouse PLFs. Lungs of older HIV-1 transgenic mice contain higher hydroxyproline content and their PLFs have a striking increase in α-SMA expression. These results suggest a mechanism by which individuals with HIV are at increased risk of developing pulmonary fibrotic changes as they age.

Change over time of the mutagenicity in the lungs of gpt delta transgenic mice by extract of airborne particles collected from ambient air in the Tokyo metropolitan area

BackgroundPreviously we found that DNA adducts were accumulated in the lungs of the rats exposed to ambient air in the Tokyo metropolitan area. To examine chronological change in in vivo mutagenicity of airborne particles, extracts produced from samples of total suspended particulates (TSP) collected from urban air in 1980, 1990, and 2010 in the Tokyo metropolitan area were intratracheally administered into the lungs of gpt delta mice, and differences in mutation and mutant frequency were determined by using the gpt assay. In vivo mutations induced by the extracts were characterized and mutation hotspots were identified by DNA sequencing of the mutated gpt gene.ResultsAdministration of the 1990 extract at a dose of 0.3 mg/animal significantly elevated total mutant frequency to 3.3-times that in vehicle control, and the in vivo mutagenicity of the extract (induced mutation frequency per milligram extract) was estimated to be 2.0- and 2.4-times higher than that of the 2010 and 1980 extract, respectively. G-to-A transition was the most common base substitution in the vehicle control mice. However, administration of the 1990 extract increased the frequency of G-to-T transversion, which is a landmark base substitution induced by oxidative stress; furthermore, when the extract was administered at a dose of 0.15 mg, the mutant and mutation frequencies of G-to-T transversion were significantly increased to frequencies comparable with those of G-to-A transition. Similar increases in the mutant and mutation frequencies of G-to-T transversion were observed after administration of the 2010 extract. Hotspots (mutation foci identified in three or more mice) of G-to-A transition mutations at nucleotides 64 and 110 were induced by the 1980, 1990, and 2010 extracts; a hotspot of G-to-T transversions at nucleotide 406 was also induced by the 2010 extract. Previously, we showed that diesel exhaust particles or their extract, as well as 1,6-dinitropyrene, administered to mice induced these hotspots of G-to-A transitions.ConclusionsThe results of the present study suggested that mutagenesis induced by extracts produced from TSP collected in the Tokyo metropolitan area induced in vivo mutagenicity via the same mechanism underlying the induction of in vivo mutagenicity by components of diesel exhaust.

Elevated Circadian Period 2: A Missing Beneficial Factor in Sickle Cell Disease By Lowering Pulmonary Inflammation, Iron Overload and Mortality

Sickle-cell disase (SCD) is a life-threatening hemolytic genetic disorder. Chronic hemolysis and elevated inflammation that underlie SCD pathophysiology is difficult to treat in the clinic due to an unclear mechanism. The role of the circadian clock is required for maintaining inflammatory states which is important for proper cellular and organ function. Circadian clocks are regulated by a series of circadian clock genes which have known functions in inflammation, heme and iron metabolism. However, the function of circadian clocks in SCD remains unknown. Here, using an unbiased and robust microarray screen, we found that genes involved in circadian rhythms, inflammatory response, heme and iron metabolism were significantly altered in the lungs of SCD Berkeley transgenic mice compared to C57BL/6 (WT) mice used as controls (N=3, P<0.05, normalized to WT). Period 2 (Per2) gene plays a role in regulating the circadian clock. Since the role of Per2 is not known in SCD, we transplanted bone marrow (BM) of SCD and WT mice to Per2Luciferase (Per2Luc) bioluminescence reporter mice to generate SCD → Per2 Luc and WT→ Per2Luc mice. Per2 circadian rhythms were determined by amplitude and periodicity in ex-vivo derived lung tissue explants. Although the period of the Per2 rhythms remained persistent throughout three circadian cycles, we demonstrated that the amplitude of circadian rhythm of Per2 was significantly upregulated in SCD mouse lung compared to WT mouse lung (N=3 per group, P<0.05). However, Per2 circadian rhythms in SCD mouse livers and kidneys did not show differences in amplitude or period compared to controls (N= 3 per group). To assess the importance of elevated Per1 and Per2 homologue genes in SCD, we transplanted BM of SCD mice to Per1/Per2 double deficient (Per1/Per2 dKO) and WT mice, respectively. To our surprise, Per1/Per2 dKO transplanted with SCD mouse BM (SCD→ Per1/Per2 dKO) displayed severe irradiation sensitivity and were more susceptible to an early death compared to WT BM transplanted mice. Mechanistically, we revealed that peripheral deletion of Per1/Per2 led to increased systemic inflammation featured with an increase of peripheral white blood cells (WBCs) in SCD→ Per1/Per2 dKO mice compared to SCD→ WT mice (50.47 ± 6.74 and 31.68 ± 1.04, respectfully, N=5 mice per group, P<0.01) without an effect on sickling in both groups of SCD chimeric mice. Moreover, we found that peripheral deletion of Per1/Per2 resulted in severe lung damage characterized by elevated congestion, neutrophil infiltration and secretion of IL-6 in lavage fluid in SCD→ Per1/Per2 dKO mouse lung compared to SCD→ WT mouse lung. Additionally, we identified that peripheral deletion of Per1/Per2 induced iron overload, heme deposits and macrophage infiltration in the lung of SCD→Per1/Per2 dKO mice compared to SCD→ WT mice. In conclusion, we discovered that Per2 is induced in SCD mouse lung but not in other organs such as kidney and liver and that its elevation is beneficial to counteract systemic inflammation, reduce pulmonary inflammatory responses, iron overload, tissue damage and thus increase survival in SCD mice. Overall, our studies reveal new molecular insight applicable to two burgeoning fields, circadian biology and SCD which pave a way for innovative therapeutic avenue for SCD. DisclosuresD'Alessandro:Omix Technologies inc: Equity Ownership; Hemanext inc: Membership on an entity's Board of Directors or advisory committees.

Rare variants in RNF213, a susceptibility gene for moyamoya disease, are found in patients with pulmonary hypertension and aggravate hypoxia-induced pulmonary hypertension in mice.

Ring finger 213 (RNF213) is a susceptibility gene for moyamoya disease (MMD), a progressive cerebrovascular disease. Recent studies suggest that RNF213 plays an important role not only in MMD, but also in extracranial vascular diseases, such as pulmonary hypertension (PH). In this study, we undertook genetic screening of RNF213 in patients with PH and performed functional analysis of an RNF213 variant using mouse models. Direct sequencing of the exons in the C-terminal region of RNF213, where MMD-associated mutations are highly clustered, and of the entire coding exons of BMPR2 and CAV1, the causative genes for PH, was performed in 27 Japanese patients with PH. Two MMD-associated rare variants (p.R4810K and p.A4399T) in RNF213 were identified in two patients, three BMPR2 mutations (p.Q92H, p.L198Rfs*4, and p.S930X) were found in three patients, whereas no CAV1 mutations were identified. To test the effect of the RNF213 variants on PH, vascular endothelial cell (EC)-specific Rnf213 mutant transgenic mice were exposed to hypoxia. Overexpression of the EC-specific Rnf213 mutant, but neither Rnf213 ablation nor EC-specific wild-type Rnf213 overexpression, aggravated the hypoxia-induced PH phenotype (high right ventricular pressure, right ventricular hypertrophy, and muscularization of pulmonary vessels). Under hypoxia, electron microscopy showed unique EC detachment in pulmonary vessels, and western blots demonstrated a significant reduction in caveolin-1 (encoded by CAV1), a key molecule involved in EC functions, in lungs of EC-specific Rnf213 mutant transgenic mice, suggestive of EC dysfunction. RNF213 appears to be a genetic risk factor for PH and could play a role in systemic vasculopathy.

An EGFR ligand promotes EGFR-mutant but not KRAS-mutant lung cancer in vivo.

EGFR ligands (e.g., EGF and TGFA) have been shown to be clinically associated with poor survival in lung cancer. Since TGFA itself initiates autochthonous tumors in liver, breast, and pancreas but not in the lung in transgenic mice in vivo, it would appear that an EGFR ligand may not initiate but rather promote lung cancer. However, it has not been proven in vivo whether lung cancer is promoted by an EGFR ligand. Using transgenic mouse models conditionally expressing EGFRL858R or KrasG12D with TGFA (an EGFR ligand) in lung epithelium, we determined that TGFA promoted the growth of EGFRL858R-lung tumors in airway regions but not that of KrasG12D-lung tumors. Analysis of TCGA datasets identified ΔNp63 and AGR2 as potential key tumor-promoting regulators, which were highly induced in the TGFA-induced EGFRL858R-lung tumors. The expression of AGR2 was positively correlated with the expression of TGFA in human EGFR-mutant lung adenocarcinomas. The expression of TGFA in human EGFR-mutant lung adenocarcinomas but not in the EGFR wild-type lung adenocarcinoma was associated with poor survival. These results suggest that targeting EGFR ligands may benefit patients who carry EGFR-mutant lung tumors but will not benefit patients with KRAS-mutant lung tumors.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli.

In patients with sickle cell disease (SCD), the polymerization of intraerythrocytic hemoglobin S promotes downstream vaso-occlusive events in the microvasculature. While vaso-occlusion is known to occur in the lung, often in the context of systemic vaso-occlusive crisis and the acute chest syndrome, the pathophysiological mechanisms that incite lung injury are unknown. We used intravital microscopy of the lung in transgenic humanized SCD mice to monitor acute vaso-occlusive events following an acute dose of systemic lipopolysaccharide sufficient to trigger events in SCD but not control mice. We observed cellular microembolism of precapillary pulmonary arteriolar bottlenecks by neutrophil-platelet aggregates. Blood from SCD patients was next studied under flow in an in vitro microfluidic system. Similar to the pulmonary circulation, circulating platelets nucleated around arrested neutrophils, translating to a greater number and duration of neutrophil-platelet interactions compared with normal human blood. Inhibition of platelet P-selectin with function-blocking antibody attenuated the neutrophil-platelet interactions in SCD patient blood in vitro and resolved pulmonary arteriole microembolism in SCD mice in vivo. These results establish the relevance of neutrophil-platelet aggregate formation in lung arterioles in promoting lung vaso-occlusion in SCD and highlight the therapeutic potential of targeting platelet adhesion molecules to prevent acute chest syndrome.

ErbB2 and p38γ MAPK mediate alcohol-induced increase in breast cancer stem cells and metastasis.

BackgroundBoth epidemiological and experimental studies suggest that excessive alcohol exposure increases the risk for breast cancer and enhances metastasis/recurrence. We have previously demonstrated that alcohol enhanced the migration/invasion of breast cancer cells and cancer cells overexpressing ErbB2/HER2 were more sensitive to alcohol exposure. However, the underlying mechanisms remain unclear. This study was designed to investigate the mechanisms underlying alcohol-enhanced aggressiveness of breast cancer. Cancer stem cells (CSCs) play a critical role in cancer metastasis and recurrence.MethodsWe evaluated the effect of chronic alcohol exposure on mammary tumor development/metastasis in MMTV-neu transgenic mice and investigated the cell signaling in response to alcohol exposure in breast cancer cells overexpressing ErbB2/HER2.Results and discussionChronic alcohol exposure increased breast cancer stem cell-like CSC population and enhanced the lung and colon metastasis in MMTV-neu transgenic mice. Alcohol exposure caused a drastic increase in CSC population and mammosphere formation in breast cancer cells overexpressing ErbB2/HER2. Alcohol exposure stimulated the phosphorylation of p38γ MAPK (p-p38γ) which was co-localized with phosphorylated ErbB2 and CSCs in the mammary tumor tissues. In vitro results confirmed that alcohol activated ErbB2/HER2 and selectively increased p-p38γ MAPK as well as the interaction between p38γ MAPK and its substrate, SAP97. However, alcohol did not affect the expression/phosphorylation of p38α/β MAPKs. In breast cancer cell lines, high expression of ErbB2 and p-p38γ MAPK was generally correlated with more CSC population. Blocking ErbB2 signaling abolished heregulin β1- and alcohol-stimulated p-p38γ MAPK and its association with SAP97. More importantly, p38γ MAPK siRNA significantly inhibited an alcohol-induced increase in CSC population, mammosphere formation and migration/invasion of breast cancer cells overexpressing ErbB2.Conclusionsp38γ MAPK is downstream of ErbB2 and plays an important role in alcohol-enhanced aggressiveness of breast cancer. Therefore, in addition to ErbB2/HER2, p38γ MAPK may be a potential target for the treatment of alcohol-enhanced cancer aggressiveness.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0532-4) contains supplementary material, which is available to authorized users.

Copper Deficiency in the Lungs of TNF-α Transgenic Mice.

Tumor necrosis factor (TNF)-α is a well-known pro-inflammatory cytokine. Increased expression of Tnf-α is a feature of inflammatory lung diseases, such as asthma, emphysema, fibrosis, and smoking-induced chronic obstructive pulmonary disease (COPD). Using a mouse line with lung-specific Tnf-α overexpression (SPC-TNF-α) to mimic TNF-α-associated lung diseases, we investigated the role of chronic inflammation in the homeostasis of lung trace elements. We performed a quantitative survey of micronutrients and biometals, including copper (Cu), zinc (Zn), and selenium (Se), in the transgenic mice tissues. We also examined the expression of Cu-dependent proteins in the inflammatory lung tissue to determine whether they were affected by the severe Cu deficiency, including cuproenzymes, Cu transporters, and Cu chaperones. We found consistent lung-specific reduction of the metal Cu, with a mean decrease of 70%; however, Zn and Se were unaffected in all other tissues. RT-PCR showed that two Cu enzymes associated with lung pathology were downregulated: amine oxidase, Cu containing 3 (Aoc3) and lysyl oxidase (Lox). Two factors, vascular endothelial growth factor (Vegf) and focal adhesion kinase (Fak), related with Cu deficiency treatment, showed decreased expression in the transgenic inflammatory lung. We concluded that Cu deficiency occurs following chronic TNF-α-induced lung inflammation and this likely plays an essential role in the inflammation-induced lung damage. These results suggest the restoration of lung Cu status as a potential strategy in both treatment and prevention of chronic lung inflammation and related disorders.

Nitrilase 1 modulates lung tumor progression in vitro and in vivo.

Uncovering novel growth modulators for non-small cell lung cancer (NSCLC) may lead to new therapies for these patients. Previous studies suggest Nit1 suppresses chemically induced carcinogenesis of the foregut in a mouse model. In this study we aimed to determine the role of Nit1 in a transgenic mouse lung cancer model driven by a G12D Kras mutation. Nit1 knockout mice (Nit1−/−) were crossed with KrasG12D/+ mice to investigate whether a G12D Kras mutation and Nit1 inactivation interact to promote or inhibit the development of NSCLC. We found that lung tumorigenesis was suppressed in the Nit1-null background (Nit1−/−:KrasG12D/+). Micro-CT scans and gross tumor measurements demonstrated a 5-fold reduction in total tumor volumes compared to Nit1+/+KrasG12D/+ (p<0.01). Furthermore, we found that Nit1 is highly expressed in human lung cancer tissues and cell lines and use of siRNA against Nit1 decreased overall cell survival of lung cancer cells in culture. In addition, cisplatin response was enhanced in human lung cancer cells when Nit1 was knocked down and Nit1−/−:KrasG12D/+ tumors showed increased sensitivity to cisplatin in vivo. Together, our data indicate that Nit1 may play a supportive role in the modulation of lung tumorigenesis and represent a novel target for NSCLCs treatment.

Abstract B22: Acyl-CoA synthetase long-chain family member 3 dependent lipid homeostasis is required for mutant KRAS driven lung cancer

Abstract Lung cancer is the leading cause of cancer related deaths in the USA and worldwide. Lung tumorigenesis is a multistep process that involves several genetic aberrations. Activating mutations of the proto-oncogene KRAS (mutant KRAS) occur in ~30% of the cases of human non-small cell lung cancer (NSCLC), which is associated with aggressive, therapy-resistant disease. Despite the recent discovery of low affinity inhibitors, mutant KRAS is a challenging therapeutic target and there is a dearth of therapeutic options for these tumors. Mutant KRAS not only promotes tumorigenesis but also the survival of established lung cancer, both in mouse models and in certain human NSCLC lines. Therefore, in the absence of clinically-relevant effective inhibitors of mutant KRAS, there has been an intense clinical interest in the development of inhibitors of its downstream effectors. Importantly, mutant KRAS cancer cells undergo oncogene-directed metabolic reprogramming in order to meet the energetic and biosynthetic challenges of cell survival, growth and proliferation. Activation of certain pathways of fatty acid synthesis has been observed in many cancer types including lung cancer. Till date, fatty acid synthase (FASN) has been the candidate for drug development. Unfortunately, the inhibitors against FASN have poor pharmacokinetics and target related toxicity concerns. There is an urgent need for discovery of additional targets that inhibit lipid metabolism specifically in cancer cells that could be exploited for therapeutic gain. The goal of our study was to identify the cellular networks that mediate the maintenance of mutant KRAS lung cancer which further could be used as high priority therapeutic targets. To this end, we functionally analyzed the transcriptome of transgenic mouse lung tumors manipulated in vivo to undergo mutant Kras extinction, providing isogenic comparisons between mutant KRAS extinguished versus non-extinguished tumors for the discovery of new therapeutic targets. We determined that mutant KRAS controls tumor metabolism by regulating lipid homeostasis. We found that Acyl-CoA synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrate for lipid synthesis and β-oxidation, is required for the survival of mutant KRAS lung cancer cells. These effects were not due to generalized toxicity, since we did not observe them in immortalized human bronchoalveolar cells and several NSCLC cells expressing wild type KRAS and in Acsl3 null mouse embryonic fibroblasts. We confirmed that ACSL3 is a mutant KRAS responsive gene expressed in the respiratory epithelium, in lung cancer cells and in primary human cancers. With mechanistic experiments we determined that mutant KRAS stimulates, in an ACSL3-dependent manner, the uptake and retention of fatty acids by lung cancer cells as well as their β-oxidation. As predicted by these experiments, ACSL3 is essential for the ability of mutant KRAS human lung cancer cells to form colonies in soft agar or to establish xenografts in immunocompromised mice. In addition, our preliminary results shows reduced tumor size and tumor burden in KrasG12D;Acsl3-/- mice. The detailed characterization of these mice is currently in progress. Our data demonstrate that mutant KRAS reprograms lipid homeostasis in lung cancer, establishing a cancer specific metabolic vulnerability. Thus, ACSL3 could be a viable therapeutic target for NSCLC driven by mutant KRAS. Citation Format: Mahesh S. Padanad, Georgia Konstantinidou, Chendong Yang, Margherita Melegari, Niranjan Venkateswaran, Kimberly Batten, Kenneth E. Huffman, Jerry W. Shay, John D. Minna, Ralph J. DeBerardinis, Pier P. Scaglioni. Acyl-CoA synthetase long-chain family member 3 dependent lipid homeostasis is required for mutant KRAS driven lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B22.

Regulation of Retinoic Acid Receptor Beta by Interleukin-15 in the Lung during Cigarette Smoking and Influenza Virus Infection.

Virus-induced exacerbations often lead to further impairment of lung function in chronic obstructive pulmonary disease. IL-15 is critical in antiviral immune responses. Retinoic acid (RA) signaling plays an important role in tissue maintenance and repair, particularly in the lung. We studied RA signaling and its relation to IL-15 in the lung during cigarette smoke (CS) exposure and influenza virus infection. In vivo studies show that RA signaling is diminished by long-term CS exposure or influenza virus infection alone, which is further attenuated during infection after CS exposure. RA receptor β (RARβ) is specifically decreased in the lung of IL-15 transgenic (overexpression; IL-15Tg) mice, and a greater reduction in RARβ is found in these mice compared with wild-type (WT) mice after infection. RARβ is increased in IL-15 knockout (IL-15KO) mice compared with WT mice after infection, and the additive effect of CS and virus on RARβ down-regulation is diminished in IL-15KO mice. IL-15 receptor α (IL-15Rα) is increased and RARβ is significantly decreased in lung interstitial macrophages from IL-15Tg mice compared with WT mice. In vitro studies show that IL-15 down-regulates RARβ in macrophages via IL-15Rα signaling during influenza virus infection. These studies suggest that RA signaling is significantly diminished in the lung by CS exposure and influenza virus infection. IL-15 specifically down-regulates RARβ expression, and RARβ may play a protective role in lung injury caused by CS exposure and viral infections.

Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is expressed in the epithelium of various primate tissues, including lung airway and alveoli. In human lung, CEACAM6 is developmentally and hormonally regulated, protects surfactant function, has anti-apoptotic activity and is dysregulated in cancers. We hypothesized that alveolar CEACAM6 expression increases in lung injury and promotes cell proliferation during repair. Studies were performed in CEABAC transgenic mice-containing human CEACAM genes. The level of CEACAM6 in adult CEABAC lung was comparable to that in human infants; expression occurred in epithelium of airways and of some alveoli but rarely co-localized with markers of type I or type II cells. Ten days after bleomycin instillation, both the number of CEACAM6(+) cells and immunostaining intensity were elevated in injured lung areas, and there was increased co-localization with type I and II cell markers. To specifically address type II cells, we crossed CEABAC mice with animals expressing EGFP driven by the SP-C promoter. After bleomycin injury, partially flattened, elongated epithelial cells were observed that expressed type I cell markers and were primarily either EGFP(+) or CEACAM6(+). In cell cycle studies, mitosis was greater in CEACAM6(+) non-type II cells versus CEACAM6(+)/EGFP(+) cells. CEACAM6 epithelial expression was also increased after hyperoxic exposure and LPS instillation, suggesting a generalized response to acute lung injuries. We conclude that CEACAM6 expression is comparable in human lung and the CEABAC mouse. CEACAM6 in this model appears to be a marker of a progenitor cell population that contributes to alveolar epithelial cell replenishment after lung injury.

Over‐expression of prothymosin‐α antagonizes TGFβ signalling to promote the development of emphysema

Emphysema, a major consequence of chronic obstructive pulmonary disease (COPD), is characterized by the permanent airflow restriction resulting from enlargement of alveolar airspace and loss of lung elasticity. Transforming growth factor-β (TGFβ) signalling regulates the balance of matrix metalloproteinase (MMP)/tissue inhibitor of matrix metalloproteinase (TIMP) to control matrix homeostasis. Patients with COPD have dysregulated TGFβ signalling and reduced histone deacetylase (HDAC) activity through epigenetic up-regulation of histone acetylation in the promoters of pro-inflammatory genes. However, the potential link between decreased HDAC activity and dysregulated TGFβ signalling in emphysema pathogenesis remains to be determined. Prothymosin α (ProT), a highly conserved acidic nuclear protein, plays a role in the acetylation of histone and non-histone proteins. The aim of this study was to test the hypothesis that ProT inhibits TGFβ-Smad signalling through Smad7, thereby contributing to emphysema pathogenesis. We show that ProT enhances Smad7 acetylation by decreasing its association with HDAC and thereby down-regulates TGFβ-Smad signalling. ProT caused an imbalance between MMP and TIMP through acetylated Smad7 in favour of MMP expression. In addition to interfering with R-Smad activation and targeting receptors for degradation in the cytoplasm, acetylated Smad7 potentiated by ProT competitively antagonized binding of the pSmad2/3-Smad4 complex to the TIMP-3 promoter, resulting in reduced TIMP-3 expression. These effects were detected in ProT-over-expressing cells, lungs of ProT transgenic mice displaying an emphysema phenotype and in emphysema patients. Importantly, increased Smad7 and reduced TIMP-3 were found in the lungs of emphysema patients and mice with cigarette smoke extract (CSE)-induced emphysema. Such effects could be abrogated by silencing endogenous ProT expression. Collectively, our results uncover acetylated Smad7 regulated by ProT as an important determinant in dysregulated TGFβ signalling that contributes to emphysema pathogenesis.

Therapeutic Effects of α1-Antitrypsin on Psedumonas aeruginosa Infection in ENaC Transgenic Mice.

Cystic fibrosis (CF) is a genetic disease with many airway pathological features, including aberrant epithelial sodium channel (ENaC) function, persistent Pseudomonas aeruginosa (PA) infection and neutrophil-dominant inflammation. PA infection in CF airways is difficult to treat due to antibiotic resistance and other factors. Recently, α1-antitrypsin (A1AT) have been shown to be effective to reduce CF airway PA infection. However, there is a dearth of studies about the mechanisms underlying A1AT’s therapeutic effects. The goal of our study is to provide an animal model of A1AT therapy in CF lungs. ENaC transgenic mice with PA infection were used as a CF-like model. Mice were intratracheally treated with PA or saline (control) in a fibrin plug. Two hours after PA infection, aerosolized A1AT were delivered to mouse lungs once daily. At day 1 and day 3 post PA infection, lung inflammation, PA load as well as host defence protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) were measured. At day 1 post PA infection when A1AT was delivered once to ENaC transgenic mouse lungs, A1AT did not reduce lung inflammation (e.g., neutrophils) and PA load. However, at day 3 post PA infection when ENaC transgenic mice received three repeated A1AT treatments, a significant decrease in airspace inflammation and PA load was observed. Although A1AT prevented the loss of SPLUNC1 in bronchoalveolar lavage fluid of PA-infected wild-type mice, it did not restore SPLUNC1 levels in ENaC transgenic mice. Our current study has provided a valid and quick A1AT therapeutic model in CF-like lungs that may serve as a platform for future mechanistic studies about how A1AT exerts beneficial effects in human CF patients.